Additional file 4: Figure S4. of Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

Mutant CALR grants factor-independence, protects from apoptosis, and activates downstream signaling in an MPL-dependent manner. a A proliferation assay was performed with the indicated cell lines (2 × 105 cells/ml). 32D MPL cells were counted every 24 h for 4 days. The cell counts are mean values of triplicates. b Empty vector, WT CALR, CALR del52, and ins5 expressing 32D cells were seeded in a density of 5 × 105 cells/ml and grown for 48 h in WEHI-free medium. Apoptosis was analyzed by flow cytometry after staining with Annexin V-APC and 7-AAD. Mean and SD are indicated. *P < 0.05, **P < 0.01, ***P < 0.001. c The indicated 32D cell lines were stably transduced with the EPOR and a proliferation assay was performed (2 × 105 cells/ml). The cells were counted every 24 h for 4 days. The cell counts are mean values of triplicates. d After 18 h starvation the indicated 32D cell lines were stimulated for 15 min with 1 U/ml EPO and lysates were prepared. SDS-PAGE, Western blotting and immunostaining with the indicated antibodies was performed. GAPDH served as loading control. e Stably transduced 32D cells with the indicated CALR-flag constructs +/− MPL were starved for 16 h and stimulated with 20 ng/ml human TPO for 15 min. Lysates were prepared and subjected to SDS-PAGE and immunoblotting using antibodies against phospho-STAT5, phospho-ERK1/2, phospho-STAT3, ERK1/2, STAT5, STAT3, and mutated CALR (CALR mut). GAPDH served as loading control. (PDF 172 kb)